Project Summary The cochlea is the organ in the auditory system responsible for the detection of sound. It houses a spiral- shaped sensory epithelium containing mechanosensory hair cells that transduce sound waves into neuronal signals. This sensory epithelium is tonotopically organized such that it detects high frequency sounds at the base of the spiral and low frequency sounds at the apex. The tonotopic specializations include a graded increase in hair cell size from the base to the apex, as well as graded changes in the electrophysiological properties of hair cells and neurons. However, the extrinsic and intrinsic factors controlling cochlear tonotopic specialization are largely unknown. The main aim of this proposal is to characterize the role of the LIN28B/let-7 axis in tonotopic specialization of auditory hair cells. The pro-growth RNA-binding protein LIN28B and the functionally opposing let-7 family of miRNAs regulate the expression of hundreds of genes, including genes involved in metabolism and cell growth. We have previously shown that LIN28B and let-7 miRNAs are expressed in opposing gradients that inform sensory progenitor cell cycle exit and hair cell differentiation. Our preliminary data show that during cochlear maturation, let-7 is highly expressed in basal outer hair cells, whereas pro-growth let-7 target genes are expressed in apical outer hair cells. Based on these findings we hypothesize that the basal-apical gradient of let-7 miRNA instructs the development of cochlear tonotopic specialization, specifically tonotopic differences in hair cell size and hair cell- specific gene expression. The development of tonotopic specialization in the mouse cochlea has not been well characterized, but it is thought that tonotopic changes start to become evident around the onset of hearing (P11). In aim 1, we propose to establish when tonotopic differences in hair cell/stereocilia size arise in the murine cochlea, by analyzing these features prior (P7), during (P14) and after the onset of hearing (P21, P30 and P60). In addition, we will characterize markers for tonotopic specialization in the murine cochlea. In aim 2, we will use existing transgenic mouse lines to abolish the gradient of let-7 miRNA in the maturing cochlea (P1- P12). At P30, we will assess the effects of these manipulations on frequency-specific hearing, as well as the tonotopic specializations of hair cells. We predict that LIN28B overexpression will produce a more `apical' identity of hair cells and functional deficits in high frequency hearing. Conversely, let-7g overexpression will lead to loss of `apical' hair cell identity and deficits in low frequency hearing. Our findings will provide insight into the molecular mechanisms regulating tonotopic identity of hair cells. This will benefit efforts in hair cell regeneration therapies, as generating appropriately specialized hair cells along the cochlea will be critical to the perception of meaningful sound.